Long Interspersed Element-1 (LINE-1 or L1) is an abundant retrotransposon that comprises ~17% of human DNA. The overwhelming majority of L1s can no longer move (i.e., retrotranspose). However, the average human genome contains ~80-100 retrotransposition-competent L1s (RC-L1s) and their mobility, in both germ line and somatic cells, has resulted in a variety of genetic disorders, including Hemophilia A, muscular dystrophy, and colon cancer. The proteins encoded by RC-L1s also likely are responsible for the mobilization of Alu elements, certain Short Interspersed Elements (SINEs) and the formation of processed pseudogenes, which together comprise ~13% of human DNA. Thus, either directly or by the promiscuous mobilization of cellular RNAs, L1-mediated retrotransposition events are responsible for at least one billion bases in human DNA and have had a tremendous impact on human genome evolution. Recent studies indicate that L1 retrotransposition events can occur during early embryogenesis and in neural progenitor cells. We hypothesize that hES cells represent a developmentally relevant in vitro model system to study the mechanisms by which L1- mediated retrotransposition events impact genome integrity at various stages of human development. An understanding of the basic molecular processes that impact genome integrity in hES cells is important to evaluate since these cells ultimately may be used therapeutically to treat a wide-range of diseases. The specific aims of this proposal are: 1. To determine the impact of L1 retrotransposition events in hES cells. 2. To determine the constellation of endogenous L1s that are mobilized in hES cells. 3. To determine how L1 retrotransposition impacts neuronal differentiation. Lay Narrative: LINE-1 elements are DNA sequences that can "jump" from one area of a chromosome into another by a process called retrotransposition. They comprise ~17% of the human DNA and have played a major role in genome evolution. The vast majorities of LINE-1 elements are mutated and can no longer jump;however, ~100 LINE-1 elements are still able to mobilize. Deleterious LINE-1 insertions into genes have caused diseases such as Hemophilia A, muscular dystrophy, and also are implicated in tumorigenesis. The proteins encoded by LINE-1 elements also are responsible for the mobilization of Alu elements, certain Short Interspersed Elements (SINEs) and the formation of processed pseudogenes, which together comprise at least 13% of human DNA. Thus, either directly or by the promiscuous mobilization of cellular RNAs, L1-mediated retrotransposition events are responsible for at least one billion bases in human DNA and have had a tremendous impact on human genome evolution. Despite these findings, it still is unclear how often, in what cell types, and when during development L1 retrotransposes in vivo. Recent studies indicate that L1 retrotransposition events can occur during early embryogenesis and in neural progenitor cells. We hypothesize that hES cells represent a developmentally relevant in vitro model system to study the mechanisms by which L1-mediated retrotransposition events impact genome integrity at various stages of human development. An understanding of the basic molecular processes that impact genome integrity in hES cells is important to evaluate since these cells ultimately may be used therapeutically to treat a wide-range of diseases.